&#9;Macrophages have evolved the capacity to recognize conserved structural componentsof LPS to facilitate effective anti-microbial responses to Gram negative bacterial infections. The objective of this research is to understand how LPS-elicited gene regulation is mediated by intracellular events. Because many LPS-regulated genes contain cAMP responsive elements (CRE) in their promoters, the cAMP response element binding protein, CREB, is likely to be important in LPS signaling. The activity of CREB is thought to be governed by phosphorylation on serine-133, and LPS treatment of macrophages activates phosphorylation of S133. The proposed research tests the hypothesis that LPS regulates CREB phosphorylation on S133, thus altering the capacity of CREB to activate transcription of CRE-containing LPS-responsive genes (LRE). Three specific aims will test this hypothesis. 1) Assess the effect of CREB phosphorylation on i) association between CREB and CBP, ii) capacity of CREB to bind CRE sequences and iii) the capacity of CREB to induce transcription. Aim 2 will determine the signaling pathways that mediate phosphorylation of CREB and identify CREB kinase(s). Aim 3 will identify the CREB-dependent LPS responsive genes. The association between CREB and CBP will be examined by co-immunoprecipitation. CREB binding to CRE will be determined by EMSA. Antibodies to CREB or phosphoCREB will detect these proteins in supershift assays. CREB transcriptional activity will be determined using a transiently transfected GAL4-CREB chimera and GAL4-CAT reporter construct. Inhibitors will be used to block LPS activated signal pathways to assess the role of that pathway in phosphorylation of CREB. An in-gel kinase assay using a peptide containing S133 of CREB will identify CREB kinases in nuclear extracts. A dominant interfering allele of CREB will be used to block the wild type protein function to identify LPS responsive genes that are CREB dependent.